In the field of nanobiotechnology, particular attention has been focussed on biochips, such as DNA chips and protein chips as an effective means for simplifying nucleic acid and protein testing in areas such as clinical diagnosis and drug development. Biochips, which are often also referred to as micro arrays, are substrates formed from glass, silicon, plastic, metal or the like on which multiple differing probes composed of bio molecules such as DNA and proteins are placed as spots in high-density areas. Binding of target molecules with probe molecules is traditionally detected by means of a fluorescence label or the like associated with the target molecules.
The present inventors have recently introduced a chip-compatible scheme for the label-free detection of bio molecules by a surface-based molecular dynamics measurement, which the inventors termed “switchSENSE method” for reasons that will become apparent below. In this method, the probe molecule is a charged molecule, particularly a charged polymer or a charged nanowire, that has a first portion attached to a substrate. The probe molecule has a marker allowing to generate signals indicative of the distance of a second portion, e.g. the distal end of the probe molecule from the substrate. The probe molecule further has a capture part capable of binding with certain target molecules that are to be detected.
In the switchSENSE method, the probe molecule is subjected to an external AC field. Since the probe molecule is charged, depending on the current polarity of the external field, said second portion of the probe molecule which is not directly attached to the substrate will approach or move away from the substrate. The change of configuration can be thought of as a switching between a “standing” configuration, in which the second portion is maximally removed from the substrate, and a “lying” configuration, in which the second portion is closest to the substrate. However, since the probe molecule is not limited to any specific shape, this terminology is rather metaphorical and should not be understood to impose any restriction on the type or shape of the probe molecule used.
By analyzing the switching behaviour between the standing and lying configurations, it is possible to detect the presence of a target molecule bound to the probe molecule. Importantly, for this detection it is not necessary that the target molecule itself is labelled in any sense, which is why the switchSENSE method is referred to as a “label-free detection method”.
For a better illustration of the switchSENSE method, reference is made to FIGS. 1 to 3, which have been taken from Ulrich Rant et. al., “Detection and Size Analysis of Proteins with Switchable DNA Layers”, Nano Letters 2009, Vol. 9, Nr. 4, 1290-1295 (prior art document 1) co-authored by some of the present inventors, which is included into the present disclosure by reference. It is to be understood that reference to this work is only meant to explain the relevant prior art but is not intended to limit the invention in any way.
In prior art document 1, (negatively charged) DNA molecules were used as probe molecules. In particular, synthetic 72-mer oligonucleotides were modified with a thiol (HS) to covalently tether the strands to the substrate, which was a gold surface in this prior art. The distal end of the DNA molecule was labelled with a fluorescent marker (cyanine die, Cy3). The single-stranded (ss) HS-ss DNA-Cy3 sequence was hybridized with a complementary strand that was modified with a protein binding tag (PBT), so that a double-stranded capture probe was formed.
The DNA layer thus formed was activated by an external electric field. Alternating potentials were applied in aqueous salt solution between the gold surface, acting as a work electrode, and a counter electrode. The applied bias polarizes the electrode interface, leading to the formation of Gouy-Chapman-Stern screening layer on the solution side. The resulting electric field was confined to the electrode proximity, with an extension of only a few nanometers, but was very intense with a field strength of up to 100 kV/cm even for low bias potentials of less than 1 V. Since the DNA is intrinsically negatively charged along its deprotonized phosphate backbone, the molecules align in the electric field and the DNA conformation can be switched between the abovementioned “standing” and “lying” state, depending on the polarity of the applied bias.
The switching action can be monitored by observing the fluorescence from the Cy3 fluorescence labels attached to the DNAs' upper ends. A non-radiative energy transfer from the optically excited dye to surface plasmons in the gold electrode quenches the emitted fluorescence intensity when the fluorescence marker, i.e. the upper DNA end, approaches the surface. Accordingly, the fluorescence marker is an example of a marker allowing to generate signals indicative of the distance of a second portion of the probe molecules (i.e. in this example, the distal ends) from the substrate, i.e. the gold surface.
In FIG. 1C, the probe molecule 10 used in prior art document 1 is schematically shown. As mentioned above, the probe molecule 10 is a double-stranded DNA, having a fluorescence marker (dye) 12 and a protein binding tag 14 attached to its distal end. Further, the gold substrate 16 and a voltage source 18 for biasing the gold surface 16 are schematically shown. If a negative voltage is applied to the gold surface or work electrode 16, the probe molecule 10 is repelled and pushed to the standing configuration schematically shown in FIG. 1C. Conversely, if a positive voltage is applied to the gold substrate 16, the distal end of probe molecule 10 approaches the same, i.e. the probe molecule 10 acquires the lying configuration. As is schematically indicated in FIG. 1C, due to the stiffness of the molecule, the switching between the standing and lying configurations is thought of as a rotation of the DNA around its fixed end.
With reference to FIGS. 1A-1D, and specifically FIG. 1B, the switching between the standing and lying configurations can be detected by observing the fluorescence light emitted by the marker 12. If the probe molecule 12 is in the standing configuration, the distance between the fluorescence marker 12 and the gold electrode 16 is the largest, and the fluorescence emission is not quenched. Accordingly, in this standing configuration, the detected fluorescence is the largest. Conversely, if the distal end of probe molecule 10 and hence the fluorescence marker 12 approaches the gold electrode 16, the aforementioned non-radiative energy transfer from the optically excited fluorescence marker 12 to surface plasmons in the gold electrode 16 quenches the emitted fluorescence intensity and thus leads to a decreased fluorescence intensity, see FIG. 1B. Herein, the difference in fluorescence intensity between the standing and lying configurations is referred to as “ΔF”.
As is demonstrated in the above-referenced prior art document 1, it is shown that binding of target molecules (proteins in the specific example) to the probe molecule 10 alters the attainable switching amplitude of the probe molecule layer, which is most likely caused by inducing steric interactions between neighbouring molecules. This phenomenon can be observed in FIG. 1B, where at the time 600 s, unlabelled streptavidin (SA) is added to the fluid environment and binds to the protein binding tag 14. As more and more of the SA binds with the probe molecule 10, ΔF decreases, until it reaches a plateau at approximately 1200 s. Accordingly, the modulation of ΔF serves as an indicator that a target molecule (in this case SA) has bound to the protein binding tag 14 of the probe molecule 10. Further, by observing the dynamic behaviour of ΔF, also the binding kinetics can be determined, in particular a binding or dissociation rate between the target and probe molecules, an affinity constant and a dissociation constant.
As is further demonstrated in prior art document 1, this label-free detection of a target molecules binding to the probe molecules via ΔF is extremely sensitive, with a detection limit below 100 fmol/l.
Further, the inventors of the present invention have shown that it is possible to discern information about the size of the target molecule by analyzing the frequency response of the switching dynamics, as will be explained with reference to FIG. 2 A-B taken from prior art document 1 as well. In FIG. 2A, the normalized switching amplitude ΔF as a function of biasing voltage frequency is shown for the pristine probe DNA in the upper curve and after binding of the immunoglobulin G (IgG) antibody of a sheep (lower curve). As a switching field, a sinusoidal ac bias voltage is applied to the work electrode, the frequency of which is varied. In FIG. 2A, the resulting frequency spectra are shown, which each comprise three distinct regimes:    (i) for low frequencies (<1 kHz), the DNA molecules follow the electrical excitation with maximal efficiency, leading to maximum oscillation of the probe molecule 10 and hence a maximum value of ΔF;    (ii) in an intermediate regime, the switching amplitude ΔF decays; and    (iii) in a high frequency region (>100 kHz), the probe molecule 10 cannot be driven by the applied AC potential anymore and hence ΔF vanishes.
The frequency range (ii) is of particular interest because it reflects the finite time constant of the switching process. To compare the switching dynamics of different samples, the frequency at which the amplitude ΔF has decreased to 50% of its initial value is evaluated. The frequency is called “cut-off frequency fc” in the following. As can be seen from FIG. 2, for the pristine DNA layer a cut-off frequency fc,1=18 kHz is found. After IgG (sheep) is bound to the DNA layer, one observes a pronounced decrease of the cut-off frequency, namely fc,2=11.5 kHz. Accordingly, it is seen that the binding of target molecules to the probe molecules slows down the dynamics of the switching process down.
It is believed that the slowing down of the switching process is caused by the increased hydrodynamic drag due to the attached target molecule. It is further believed that the increment in molecular weight does not play an as important role, since the DNA motion is extremely over-damped, so that inertial effects can be neglected. As has been shown by the inventors of the present invention, the frequency shift induced by target molecules can be used to determine the target molecules' size, in particular their effective Stokes radius. With regard to the example of FIG. 2, the inventors have measured frequency shifts for various antibodies and antibody fragments. FIGS. 3A and 3B summarize cut-off frequency measurements for proteins of varying size. The normalized cut-off frequency is plotted versus the normal molecular weight in panel A (Fab sheep (50 kDa), streptavidin (75 kDa), Fab2, goat (100 kDa), IgG sheep (150 kDa) and IgG goat (160 kDa)). The cut-off frequency versus the respective hydrodynamic diameters Dh are shown in panel B. FIG. 3C shows the Dh distribution measured by dynamic light scattering, see prior art document 1 for more details.
As can be seen from FIG. 3, a monotonous decrease of the normalized cut-off frequency is found for increasing size of the bound protein. In particular, Fab and Fab2 fragments can be clearly discriminated from uncleaved antibodies. Accordingly, measuring the frequency response of the fluorescence amplitude, the size of the target molecules can be evaluated with remarkable precision.
As is obvious from the above, using the switchSENSE method, both the binding of the target molecule as such as well as the size of the target molecules can be evaluated for unlabelled targets. In particular, the evaluation of target molecule size by analyzing the frequency response has proven to be a very powerful tool that requires only limited experimental effort and proved to be very robust. The frequency response analysis is also the subject of further publications and patent applications co-authored by some of the present inventors, see in particular U. Rant et. al, “Switchable DNA Interfaces for the Highly Sensitive Detection of Label-free DNA Targets”, PNAS (270), Vol. 104, Nr. 44, p. 17364-17369, EP 2 192 401 A1 and US 2005/0069932 A1.
While the combined detection and size evaluation of target molecules according to the above prior art has proven to be very successful, there is an on-going desire to increase the precision and reliability of the evaluation of characteristics of the target molecules. A further object of the invention is to provide a method for evaluating characteristics of a target molecule that would be particularly suitable for implementing in commercially available apparatuses, thus lifting the switchSENSE technology from a scientific concept to a practical tool that can be routinely used not only in academic research but also in pharma and biotech industry as well as laboratories for clinical diagnostics and hospitals. A further problem underlying the invention is to provide an apparatus that would allow evaluating characteristics of a target molecule bound to a probe molecule with good precision, yielding reliable and trustworthy results to routine users who cannot be expected to question the analysis results but instead need to rely on them.